| Summary: | Bovine mastitis is mainly caused by bacteria of the genus <i>Staphylococcus</i> spp., which possess different virulence factors, including the capacity for biofilm formation that provides enhanced protection against the action of immune system components and serves as a barrier against the penetration of antimicrobial agents. This study aimed to characterize 181 <i>Staphylococcus</i> spp. Strains—including <i>Staphylococcus</i><i>aureus</i> and coagulase-negative staphylococci (CoNS) isolated from bovine subclinical mastitis in six Brazilian states—by molecular methods. RT-qPCR was used to verify the expression of genes of the <i>ica</i> operon—mainly responsible for biofilm formation—as well as <i>bap</i> and <i>bhp</i>. Chromosome similarity among the isolates was investigated by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The <i>ica</i>A gene was detected in 79 (43.6%) isolates, <i>ica</i>B in 24 (13.2%), <i>ica</i>C in 57 (31.4%), and <i>ica</i>D in 127 (70.1%). The <i>bap</i> gene was identified in 66 (36.4%) isolates, while the <i>bhp</i> gene was found in nine (4.9%). RT-qPCR confirmed the expression of the <i>ica</i>A gene in 60 (75.9%) isolates, of <i>ica</i>B in six (25%), of <i>ica</i>C in 26 (45.6%), and of <i>ica</i>D in 80 (63%). Clonal typing of the isolates by PFGE permitted the identification of eight <i>Staphylococcus</i><i>aureus</i> clusters that simultaneously included ≥3 strains, with a similarity of ≥80%. Regarding the other species studied, three clusters were observed for <i>Staphylococcus</i><i>chromogenes</i> and four clusters for <i>Staphylococcus</i><i>epidermidis</i>. Only one cluster each was identified for <i>Staphylococcus</i><i>saprophyticus</i> and <i>Staphylococcus</i><i>simulans</i>, while the other species did not form any cluster. With respect to MLST, ST126 and ST1 were the prevalent sequence types in <i>S. aureus</i>, while in <i>S.</i><i>epidermidis</i> all sequence types were different. These results reveal strains with the same evolutionary origin as other isolates, which might cause infections in humans and animals, suggesting their ability to spread between these species.
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